Actin is involved in many cellular processes such as morphogenesis, intracellular transport, cell division, muscle contraction and cell migration. The actin cytoskeleton is also altered in disease processes such as in invading tumour cells, myopathies or polycystic kidney disease(1-3). In studying the various mentioned processes and conditions a reliable staining of the actin cytoskeleton is essential. In most cases staining of fixed cells with the F-actin binding compound phalloidin coupled to fluorescent dyes is used. However, some cells such as the plant pathogenic fungus Ustilago maydis, cannot be stained by phalloidin(4). In addition it is often desirable to image actin in living cells. The dynamics of actin filaments are much more sensitive readouts for cytoskeletal organization and processes such as cell polarization or cell migration can only be properly studied through analysis of cytoskeletal dynamics.
To study actin dynamics, researchers have relied either on the injection of fluorescently labelled actin or small amounts of phalloidin(5-6) or on the use of GFP fusion proteins. In the former case application is limited to large cells that can be injected, requires specialized equipment, relatively expensive probes and quantitative analysis is complicated by difficult control of the fluorescent actin concentration. Speckle analysis has provided a powerful tool for the detailed study of actin polymerization dynamics with only trace amounts of labelled molecules. Various GFP fusion proteins have been used to visualize actin in living cells. Most often actin itself has been fused to GFP but all documented actin-GFP fusion proteins exhibit reduced functionality and they can only be used in combination with non-tagged actin present(7). Even in cases where cells are not visibly affected by actin-GFP expression it is not certain whether actin dynamics is partially affected. Therefore, while actin-GFP can be used in several cell types to monitor actin distribution it is to be used with great care if studying actin dynamics. Actin-GFP is also limited in its application as it exhibits a strong background staining from labelled actin monomers and therefore requires low expression levels. Alternatively fusion of GFP to several actin binding domains have been used, notably from moesin in Drosophila(8), LimE in Dictyostelium(9), Abp120 in Dictyostelium and mammalian cells(10,11) and utrophin in Xenopus(12). In plants fusions to the actin binding domains of mouse talin(13) or fimbrin(14) have been used but each seems to stain only a subset of actin structures and can lead to artificial bundling of actin if expressed at high levels(15,16). In general the used fusion proteins are still quite large and are restricted to cells that can be transfected or injected.